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This flexibility may arise from inter-repeat motion Figs 2d,e and 4a,b ; Supplementary Figs 8 and 9 or the presence of a C-terminal tail including EC11 residues and a His-tag used for protein purification.

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Despite this flexibility, ab initio modeling from the experimental scattering profiles produced PCDH15 EC8—10 shapes in solution that are in remarkable agreement with crystallographic data Fig. Both the EC8—9 contortion and the EC9—10 kink can be observed in the filtered shape reconstruction showing an S-shaped molecule.

The function of tip links is to convey force and pull open inner ear transduction channels. To facilitate this process, myosin motors pull on tip links and keep a resting tension that maintains transduction channels optimally poised to respond to external mechanical stimuli 16 , The elasticity of the entire tip link is unknown, but previous studies on the elasticity of CDH23 suggest that its EC repeats are stiff and inextensible unless large forces are applied to unfold them The structure of PCDH15 EC8—10 suggests an alternate scenario where EC9—10 unbending might be relevant during force transduction: as force is applied to tip links, unbending results in protein extension without unfolding tertiary structure elasticity: TSE 43 , Protein is shown as ribbons and coloured as in Fig.

Arrows indicate position and direction of the applied forces. A similar, albeit less pronounced, lengthening event was observed in one of the equilibrium MD simulation of the structure Supplementary Fig. Similar constant-force simulations of the bent DNcad EC2—3 structure showed a different mechanical response.

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Therefore, its TSE is predicted to be relevant during mechanotransduction. To further explore the elasticity of PCDH15 EC8—10, we carried out constant-velocity SMD simulations in which both protein ends are attached to springs that are pulled in opposite directions. The observation of these two phases suggests that unbending is important for the elastic response of PCDH Protein is shown in ribbon representation and coloured as in Fig.

Springs indicate position and direction of the applied forces. Rupture of these interactions correlates with unfolding force peaks. Rupture of these interactions correlates with unbending. Unbending of PCDH15 EC9—10 during the slowest constant-velocity stretching simulation was similar to unbending in constant-force mode. At this point, the EC9—10 linker was fully extended, but a few contacts between the two EC repeats remain. These events were accompanied by a sudden drop in applied force and marked the beginning of EC10 unfolding.

A second force peak correlated with the rupture of backbone hydrogen bonds Tyr 1,—Lys 1, and Tyr 1,—Tyr 1, Fig. These residues are part of the EC9—10 linker and do not interact with each other in the native, crystallographic conformation. However, this salt bridge is intermittently formed in one of our equilibrium simulation S1b as a consequence of the relaxation of the EC9—10 3 10 helix and a conformational change of the EC10 supporting loop 1,—1, discussed above Supplementary Fig.

Formation of the Glu 1,—Arg 1, salt bridge may help prevent unfolding by strengthening the EC9—10 linker in its extended conformation. To quantify the stiffness of PCDH15 EC8—10 we computed the slope of the linear regions of the force versus end-to-end distance monitored in simulations using the slowest pulling velocities S5b at 0. Known structures, including EC8—10, are shown. Unbinding with minimal unfolding follows unbending. Constant-velocity SMD simulations of the chimeric complex using stretching speeds of 0.

Force monitored throughout these simulations Fig.

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Stretching at slower speeds of longer tip links may reveal an even smaller effective spring constant. Unfolding of EC10 required more force These simulations suggest that the PCDH15 EC9—10 linker does not unfold and may unbend before unbinding during tip-link-mediated mechanotransduction.

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The structural studies and simulations of PCDH15 presented here provide a first view into its non-canonical cadherin EC repeats and their function as part of inner-ear tip links. Multiple lines of evidence suggest that this conformation is particular and favoured in solution. First, the lack of calcium-binding motifs sequences at the EC9—10 linker is highly conserved across different species.

These features are also conserved with only subtle differences between mammalian, aves and reptilian, amphibian and actinopterygian sequences.

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Last, short and long timescale MD simulations as well as SAXS experiments strongly support a bent and somewhat flexible conformation in solution. Interestingly, the highest resolution views of tip links in situ show what could be interpreted as a bent EC9—10 linker near the point of membrane insertion of PCDH15 Fig. However, the human EC10—11 linker lacks one of the residues involved in calcium binding Supplementary Fig. Unbending transitions provide a soft elasticity that resembles TSE predicted for ankyrin repeats 43 and titin domains Small, but not negligible force is required to reversibly straighten these proteins without unfolding, a process that will affect how force is conveyed to transduction channels.

However, the spring constant estimates from simulations represent an upper boundary to physiological values, as stretching speeds in silico are significantly faster than those used in the experiments that were used to estimate the gating spring constant Supplementary Discussion. In PCDH15, the EC5—6 linker lacks calcium-binding residues and might unfold, be flexible, or adopt a bent conformation as well 33 , 38 , 45 , Also, a possible EC12 repeat and its linker to EC11 could provide additional elasticity along with the lipid membrane around the transduction channel 47 , Regardless of the mechanism, the elastic response of the whole transduction apparatus will be dominated by the softest component, yet to be determined.

Clearly, more complete structural models of the hair-cell transduction apparatus are required to dissect out the exact contribution of each of its components to the gating spring elasticity, with unbending TSE of PCDH15 EC9—10 being part of it. Tip link lengthening due to unbending could lead to decreased tip link tension and channel closure. Calcium would have to regulate this process allosterically through other PCDH15 and CDH23 EC repeats with calcium-binding motifs, thereby introducing a non-linear response and modulating the stiffness of the release element as proposed by Martin et al.

In addition to unbending, unfolding of PCDH15 EC repeats can also drastically alter how force is conveyed to transduction channels. Calcium is known to help prevent mechanical unfolding in cadherins 36 , 54 , and mutations that alter cadherin calcium-coordinating residues cause deafness see Supplementary Discussion and Supplementary Fig.

A calcium-free inter-repeat linker in PCDH15 could be prone to unfolding Simulations of an artificial chimeric model involving all published structures of tip link EC repeats suggest that unbending of EC9—10 is followed by unbinding without any relevant unfolding, as observed for other classical cadherins 55 , However, this artificial model does not take into account the effect of parallel dimerization see below and does not incorporate the behaviour of other EC repeats and atypical linkers that may unfold before unbinding occurs.

Relating the nanomechanics of tip link structures to microscopic correlates of hair-cell bundle motion and channel gating obtained from biophysical experiments is challenging 47 , 48 , Tip links are enormous proteins, and structures for many EC repeats are still missing. Parallel dimerization may further alter, perhaps in trivial ways, the mechanical response of the whole assembly Glycosylation could also modulate dimerization and elasticity of PCDH15 ref. Moreover, while sound transduction is a fast process that can occur in microseconds, SMD simulations are performed at stretching velocities that match only part of the spectrum of physiological stimuli and that may not completely capture the viscoelastic response 60 of tip link cadherins see Supplementary Discussion.

Yet our structure and simulations of PCDH15 EC8—10 points to a key mechanical component of the tip link that is elastic and that must be incorporated in hair-cell transduction models. Overall, the data presented here strongly suggest that the bent, calcium-free linker in PCDH15 EC9—10 plays a relevant mechanical role in hair-cell transduction.

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Our findings may have implications beyond PCDH15 function in the inner ear. Sequence analyses have shown that calcium-free linkers are found in multiple members of the cadherin superfamily, yet their function is unknown. Lack of a common sequence motif among these calcium-free linkers except for the absence of calcium-binding residues suggests that they could provide a variety of conformations and play diverse roles in cadherin function Perhaps some of these linkers endow non-classical cadherins with specific mechanical functions beyond the classical paradigm 59 , All crystals were cryo-cooled in liquid N 2.

X-ray diffraction data was collected as indicated in Table 1 and processed with HKL The mmPcdh15 EC9—10 structure was indexed in space group P 3 1 after detecting tertatohedral twinning. Data collection and refinement statistics are provided in Table 1. Buffer matched controls were used for buffer subtraction. Hydrogen atoms were automatically added to protein structures and crystallographic water molecules.

Residues D, E, K and R were assumed to be charged. Histidine residues were assumed neutral, and their protonation state was chosen to favour the formation of evident hydrogen bonds. These two structures where solvated and ionized as mentioned above. The resulting model for the linker was energy-minimized in vacuum 2, steps , and subsequently analysed with the CheckMyMetal server 72 to ensure proper coordination of calcium ions Supplementary Fig.

Coordinates of the entire chimeric complex are available upon request. Anton is a massively parallel special purpose machine for molecular dynamics simulations Anton and NAMD simulations used the same force field. Infrequent updates of both thermostat and barostat improve the performance of the simulation and numerical integration accuracy. In our simulation protocol, the Langevin thermostat 75 was updated every 24 steps and the MTK barostat was updated every steps.

SHAKE was used to constrain all bonds involving hydrogen atoms. Each system was energy-minimized and equilibrated in the constant number, pressure and temperature ensemble NpT , and the resulting state was used to perform subsequent equilibrium and SMD simulations Table 2. The stretching direction was set along the x axis matching the vector connecting terminal regions of the protein. The free ends of the springs were moved away from the protein in opposite directions at a constant velocity.

Applied forces were computed using the extension of the virtual springs. Plotted forces correspond to those applied to the N-terminal atoms unless otherwise stated.

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Stiffness was computed through linear regression fits of force-distance plots. Plots and curve fits were prepared with QtiPlot. Molecular images were created with the molecular graphics program VMD The remaining data that supports the findings of this study are available from the corresponding author upon reasonable request. How to cite this article: Araya-Secchi, R. An elastic element in the protocadherin tip link of the inner ear.

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